J. Med. Chem. 1985,28, 733-740 (K,CO,) and evaporating in vacuo. Crystallization of the orange free base was accomplished from 2-PrOH. The dihydrochloride salt was prepared from the base in EtOAc solution by addition of anhydrous HCl in EtzO. Ten grams (38%) of Pale Yellow needles gave mp 231-233 O C (with intumescense)* Anal. (c14H17N30y2HCl) C, H, N. 3,4,6,6-Tetrahydro-4-(2-propenyl)-2H-1,5-metha~o-l,4bnzdiazocin-gamine Diethanesulfonate Hemihydrate (12). Reduction of the nitro group of 11 was accomplished by the addition (without the application of external heat) of four 2-g portions of Fe powder to a solution of 10 g (0.03 mol) of the HCl salt Of in mL Of HoAc and lomL Of H2°* After Overnight stirring and then evaporation to dryness, the residue was partitioned between aqueous NaoH and CHC13* Evaporation Of the dried (KzC03) CHC13 left a tan powder$which was converted into the diethanesulfonate in 2-ProH* Recry'from 2-ProH gave g (7%)9 mp 21&220 OC* Of product 12. Anal. (Cl4HleN3.2CZHSSO3H"/2H~O) C, H, N.
Acknowledgment. We thank Dr. Stephen D. Clemans for interpretation of NMR protonation and chiral shift
733
reagent studies. We thank John D. Grego for GC studies of racemic and resolved la using the Masher's reagent. We we grateful to D ~william . H,Thielking, F.D ~and~ Edward D. Par& for development work resulting in improved synthetic techniques for making these compounds. Dr' David G*Teiger provided dependence, abuse liability evaluations, and EDTA antinociceptive data on 6. We thank especially Anne K. PierSon for the narcotic Wonist/antagonist bioassays. Registry No. ( i ) - l a ,80769-97-7; (-)-la, 80770-08-7; (-)la.l-mandelate, 95763-76-1; (+)-la, 80770-13-4; (+)-la.dmandelate, 95763-77-2; (*)-3a, 80769-98-8; (i)-3b, 80770-20-3; (&)-4a,80770-00-9; (&)-4b,80770-21-4; (&)-5,80770-01-0;(-&)-5a, 80770-31-6;( & ) 480770-03-2;(+)-6,80770-16-7;(-)4 80770-12-3; ( & ) 4 (diazonium sulfate), 95763-82-9; (&)-7,95763-78-3; (&)-S, 80770-30-5; (&)-9a,80770-28-1;(&)-9a.2HC1, 80770-29-2; (&)-9b, 80770-32-7; (i)-10, 80770-23-6; (&)-10*2HCl,95763-79-4; (A)ll.2HC1, 95763-80-7; (34-12, 80770-25-8; (i)-12.2CzH6S03H, 80770-26-9;cyclopropanecarboxylic acid anhydride, 33993-24-7.
Synthesis and Antimicrobial Evaluation of Bicyclomycin Analogues Robert M. Williams,*?+Robert W. Armstrong, and Jen-Sen Dung Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523. Received September 24, 1984 The synthesis and antimicrobial evaluation of novel bicyclomycin analogues are described. The series of analogues were prepared from the basic 8,10-diaza-2-oxabicyclo[4.2.2]decane-7,9-dione ( S ) , 7,9-diaza-2-oxabicyclo[3.2.2]nonane-6,8-dione (9), 8,10-diaza-5-methylene-2-oxabicyclo[4.2.2]decane-7,9-dione (lo), and 7,9-diaza-4-methylene-2oxabicyclo[3.2.2]nonane-6,8-dione(11) nuclei. For compounds where R1 = p-methoxybenzyl, deprotection of the lipophilic amides with ceric ammonium nitrate affords the corresponding lipophobic free amides. The basic bicyclic nucleus of bicyclomycin (8h, R1= Rz= R3 = R4 = H) has been synthesized for the first time as well as increasingly more complex congeners bearing the C-6 OH, 5-methylene; (3-1'4-3' trihydroxyisobutyl group. In general, it has been found that the bicyclic nucleus of bicyclomycin is devoid of antimicrobial activity, the entire structure of bicyclomycin being generally obligate for activity. In one instance, the racemic analogue 100 (R, = CH2Ph, Rz = OH, & = H) showed interesting antimicrobial activity against several Gram-positive organisms;the minimum inhibitory concentrations were of the same order of magnitude as bicyclomycin displays toward Gram-negative organisms. Totally synthetic (A)-bicyclomycin was half aa active as the natural antibiotic. The design, synthesis, and antimicrobial activity (and/or lack thereof) of bicyclomycin and the analogues are discussed in the context of a proposed chemical mechanism of action.
Bicyclomycinl (bicozamycin, 1) is an antibiotic obtained from cultures of S t r e p t o m y c e s sapporonensis2 and Streptomyces a i ~ u n e n s i s . ~ Bicyclomycin is biosynthesized4from leucine and isoleucine and possesses a unique chemical structure amongst the known classes of antibiotics. The low toxicity and ready availability of biA
Scheme I
O 'H
I
O 'H
-2
O 'H
-3
Bicyclomycin is active against Gram-negative bacteria such as Escherichia coli, Klebsiella, Shigella, Salmonella, HO+M~
-I cyclomycin from fermentation have resulted in the recent commercial introduction of bicozamycin6 as an effective agent against nonspecific diarrhea for humans and bacterial diarrhea of calves and pigs.6 The mechanism of action of bicyclomycin is also thought to be unique and has been the subject of several accounts.' NIH Research Career Development Awardee 1984-1989.
(1) (a) Miyoshi, T.; Miyairi, N.; Aoki, H.; Kohsaka, M.; Sakai, H.;
Imanaka, H. J. Antibiot. 1972,25,569. (b) Kamiya, T.; Maeno, S.; Hashimoto, M.; Mine, Y. Ibid. 1972,25, 576. (c) Nishida, M.; Mine, Y.; Matsubara, T. Ibid. 1972,25, 582. (d) Nishida, M.; Mine, Y.; Matsubara, T.; Goto, S.; Kuwahara, S. Ibid. 1972, 25, 594. (e) Miyamura, S.; Ogasawara, N.; Otsuka, H.; Niwayama, S.; Tanaka, H.; Take, T.; Uchiyama, T.; Ochiai, H.; Abe, K.; Koizumi, K.; Asao, K.; Matsuki, K.; Hoshino, T. Ibid. 1972, 25,610. (f) Miyamura, S.; Ogasawara, N.; Otsuka, H.; Niwayama, S.; Tanaka, H.; Take, T.; Uchiyama, T.; Ochiai, H. Ibid. 1973, 26, 479. (2) References la-d. (3) References l e and If. (4) (a) Miyoshi, T.; Iseki, M.; Konomi, T.; Imanaka, H. J. Antibiot. 1980, 33, 480; 1980, 33, 488.
0022-2623/85/1828-0733$01.50/0 0 1985 American Chemical Society
,
734 Journal of Medicinal Chemistry, 1985, Vol. 28, No. 6
Scheme I1
Williams, Armstrong, Dung Scheme 111 Nu
Nu
‘OH
HO-/-Me
-4
‘OH
-7 Scheme IVa HO HO-&Me ‘OH
O ‘H
-6 Citrobacter, Enterobacter cloacae, and Neisseria but is inactive against Proteus, Pseudomonas aeruginosa, and Gram-positive bacteria. Iseki et d.’have shown that bicyclomycin irreversibly and covalently binds inner-membrane proteins (BBP’s) of E. coli that are distinct from penicillin-binding proteins. The function of the bicyclomycin-binding proteins (BBP’s) and the chemical mechanism by which bicyclomycin binds to these proteins remains to be determined. The stoichiometry of the bicyclomycin-BBP complex has been shown7to be 1:1, and further, the binding is inhibited by the addition of thiols.8 A Ciba-Geigy groupg has prepared a large number of semisynthetic bicyclomycin derivatives and found that most structural modifications result in reduction or loss of biological activity. Unfortunately, a clear structureactivity relationship has not emerged from the abovementioned studies. Iseki and co-workers’ reported on the reaction of the C-5 exo-methylene moiety of bicyclomycin with methanethiol at high pH and suggested that the C-5 double bond may be the active functionality that could irreversibly alkylate a sulfhydryl residue on the BBP.8 Careful inspection of the bicyclomycin structure and consideration of the regiochemistry of the mercaptan addition suggest that bicyclomycin may act as a “latent” &-unsaturated pyruvamide (2), which should undergo facile Michael-type addition at C-5=CH2 (1 2 3, Scheme I). Such a general-base-catalyzed mechanism readily accounts for the regiochemistry of the mercaptan adduct (3, R = CHJ reported by Iseki et al.7 Alternatively, bicyclomycin may irreversibly alkylate the BBP’s by a distinctly different but related “latent” Michael-acceptor mechanism in vivo. Being itself a peptide, bicyclomycin may be interacting with a protease or transpeptidase type protein that functions by catalytically cleaving a peptide bond during the biosynthesis of the bacterial cell envelope. As proposed in Scheme 11, cleavage of the 9,lO-amide bond by the protein produces acyl en-
--
Merck Index, 10th ed., No. 1213. Bicozamycin is the commercial name licensed to Fujisawa Pharmaceutical Co., Japan; aizumycin and bicyclomycin are synonyms; “bicyclomycin” shall be used throughout this paper. Private communication, Fujisawa Pharmaceutical Co., Japan. (a) Someya, A.; Iseki, M.; Tanaka, N. J . Antibiotics 1978,31, 712. (b) Tanaka, N.; Iseki, M.; Miyoshi, T.; Aoki, H.; Imanaka, H. Ibid. 1976, 29, 155. Someya, A.; Iseki, M.; Tanaka, N. J . Antzbiot. 1979, 32, 402. A detailed mechanism for the thiolate addition has not been suggested. Muller, B. W.; Zak, 0.; Kump, W.; Tosch, W.; Wacker, 0. J . Antibiot. 1979, 32.
16 -
17 -
18 -
a ( a ) 2 equiv of LiAlHJTHF, 25 ”C, 1 min; Na,SO,~lOH,O quench; ( b ) 1equiv of AgClO,, THF, 25 “C; (c) MsC1, Et,N, THF; (d) NaBH,SePh, THF; (e) 30% H,O,, THF, 55 “C; ( f ) 0,, CH,Cl,; (g) NaBH,, MeOH.
zyme derivative 4. The amide-derived NH2 (at C-6, 4) should be rapidly expelled as NH3loat physiological pH, forming the cu,@-unsaturatedpyruvamide 5a, which may similarly undergo conjugate addition resulting in the “suicide” inactivation of the BBP. Finally, it is possible that a nucleophile may undergo allylic addition at C-5 that does not per se require the intermediacy of a ring-opened a,P-unsaturated species such as 2 or 5 (Scheme 111, 1 7). In order to gain some insight into the chemical mechanism of action of bicyclomycin, vis-a-vis the proposals outlined above, we have designed and synthesized a series of bicyclomycin analogues and have evaluated them for biological activity. We were primarily interested in probing the obligate partnership of the C-5 exo-methylene moiety and the C-6 hydroxyl group as a minimal structural requirement for biological activity rather than making more random peripheral structural changes. Furthermore, all of our synthetic analogues were specifically prepared in racemic form to allow for the greatest versatility in identifying intrinsic biological activity; all previously reported semisynthetic analogues possessed only the natural configuration. Recently, we have re~0rtedll-l~ on the development of a versatile and efficient synthesis of the bicyclo[4.2.2] nucleus (8 and 10, R, = R3 = H) that can be regioselec-
-
Depending upon the localized pH, it is also possible that the C-6 OH is lost as H20from 4, forming the corresponding a$unsaturated imino pyruvamide 5b that may either hydrolyze to 5a or directly undergo conjugate addition. Williams, R. M.; Anderson, 0. P.; Armstrong, R. W.; Josey, J.; 1982, 104, 6092. Meyers, H.; Eriksson, C. J. Am. Chem. SOC. Williams, R. M.; Dung, J.-S.; Josey, J.; Armstrong, R. W.; Meyers, H. J. Am. Chem. SOC. 1983, 105, 3214. Armstrong, R. W.; Dung, J.-S.; Williams, R. M. 185th National Meeting of the American Chemical Society, Division of Organic Chemistry, Seattle, WA, March 1983; Abst. 10. Dung, J.-S.;Armstrong, R. W.; Williams, R. M. J. Org. Chem. 1984, 49, 3416.
Journal of Medicinal Chemistry, 1985, Vol. 28, No. 6 735
Bicyclomycin Analogues Scheme V
nA
0 4
HO
5 Bu~NF
O ‘H
2 3 1
12b
19
36 %
OA I. TFA A / CH2CI2
2. CAN /Me CN/H20
3 BuLi
4. Bn= CH2
CHO
HO HO+Me
-
35 %
2: I
4 0%
12c -
20 -
tively elaborated into a multitude of structurally diverse bicyclomycin analogues via carbanion substitution at the C-1 and C-6 bridgehead positions. This methodology has allowed making deepseated functional group modifications in the bicyclo[4.2.2] nucleus from a few simple bicyclic precursors. In increasing order of complexity, we have examined analogues both bearing and lacking the following: (a) substitution of N-8 and N-10, (b) the C-6 hydroxyl group, (c) the C-5 exo-methylene moiety, and (d) the C1°C-3’ trihydroxyisobutyl side chain. In addition, we have developed a parallel series of analogues based on the homologous bicyclo[3.2.2] ring system (9 and 11)whose anticipated increased ring strain was hoped to impart increased biological activity. Results and Discussion The bicyclic precursors 8 and 9 (R, = alkyl, aryl; Rz = R3 = R4 = H) were prepared as previously des ~ r i b e d . ~ ~ JSubstrates ~ J ~ J ~ 10 were prepared according to the methods we have recently d i s ~ l o s e d l ~in*our ~ ~ ,total synthesis of bicyclomycin. The homologous bicyclo[3.2.2]nucleus 11 was prepared as outlined in Scheme IV. The syn lactone 14 or its syn diastereomer could be converted into bicyclic olefin 18 by the procedure that is outlined. It is significant to point out that the bicyclo[3.2.2] nucleus 18 contains considerable strain energy as evidenced by the 1695-cm-’ infrared absorbtion of the amide carbonyls relative to the bicyclo[4.2.2] nucleus 10a (1660-1675 cm-l). These common bicyclic nuclei 8-11 (R, = R3 = H, Chart I) served as the substrates from which the analogues were regioselectivity elaborated according to the protocol we have developed.l1JZ Table I provides a tabulation of the compounds synthesized that have been evaluated for antimicrobial activity. It is significant to point out that, of the amide N-protecting groups we have evaluated (CH,, CH20CH3,CH2Ph, (15) Satisfactory combustion analytical data were reported for 8
and 9. (16) (a) Williams, R. M.; Armstrong, R. W.; Dung, J.4. J. Am. Chem. SOC.1984,106, 5748 and references cited therein. (b) Williams, R. M.; Armstrong, R. W.; Dung, J.-S. J. Am. Chem. SOC.,in press.
O ‘H
Chart I
-8
~ ~ 0 ’ ~ ’
12 -
-9
~~0’3’
13 -
-
Ph-p-OCH3, CH,Ph-p-OCH,), only the N-(p-methoxybenzyl) amides could be deprotected (N-R N-H) under sufficiently mild conditions (ceric ammonium nitrate (CAN), MeCN, H20, 25 “C) for the bicyclic structure and attendant functionality to remain intact. The identification of a suitable and generally useful blocking group for the amides turned out to be a crucial element for the present study as well as our related studies on the total synthesis of bicycl~mycin.~~J~ The corresponding N-benzyl protecting group, which has been utilized in a recently ~ornmunicatedl~ total synthesis, was found to be uniformly unsuitable for making bicyclic structures bearing free-NH (17) Nakatsuka, S.; Yamada, K.; Yoshida, K.; Asano, 0.; Murakami, Y.; Goto, T. Tetrahedron Lett. 1983, 24, 5627.
736 Journal of Medicinal Chemistry, 1985, Vol. 28, No. 6 Table I. Synthetic Bicyclomycin Analogues Submitted for Antimicrobial Assay' compd R4 rep R1 RZ R3 8a CH, H H H 11 8b CHiPh H H H 11 CHZPh-p-OCH3 H H H 14 8c Ph-p-OCH3 H H H 14 8d H 8e 12 SCH3 H CH3 8f 12 SCH3 H CH3 CH3 H 12 CH3 CHOHPh H 8g 8h H H H H C H OH 8i H H C CHzPh OH H H C 8j 9a H H 12 CH3 H 12 9b H H COPh CH3 H H H CH=CHz C 9c CHzPh H H CH=CH2 C 9d CHzPh H H CHzOH C 17 H H 16 1Oa CHzPh H H 10b H b OH H 16 1oc CHzPh OH H C 10d H 18 C CHzPh H H 12a CHZPh C OH (Me)& 12b H H H H C 12c H OH H H C OH H H 13a H 16 "See Table I1 for the standard 20 microorganism assay used. See indicated reference for experimental preparation. e New compound not previously reported; experimental procedure appears in the Experimental Section.
amides. In our hands, numerous oxidative (CAN, Cr03, DDQ), reductive (Li/NH,, Na/NHB, 10% Pd/C, 20% Pd/C, 20% Pt(OH),/C), and hydrolytic (H3P04/PhOH, TFA, HBr) attempts (many solvents, temperatures, etc. examined) to remove the N-benzyl groups on more than a dozen bicyclic compounds resulted in cleavage of the bicyclic system (C-1-0 ether linkage) and/or (in the case of hydrogenolysis) saturation of the benzylic aromatic rings. Scheme V illustrates the synthesis of demethylenebicyclomycin 12c and 6-deoxydemethylenebicyclomycin 12b from the same unsubstituted bicyclic precursor 8c. Both systems displayed relatively modest selectivity in the aldol condensation step as compared to that we have achieved16in the total synthesis of 1. This was especially true for the deoxy compound 19,which was the intermediate diastereomer (shown) in the condensation.lg The anomalous behaviorl1J2J6of this aldol condensation may be attributable to the C-6 trimethylsilyl residue, which presumably alters the conformation of the bicyclic carbanion relative to the C-6 alkoxy species. As expected, removal of the amide blocking groups changes the solubility characteristics of the structure from being geqerally lipophilic/ hydrophobic (N-alkylated) to hydrophilic/lipophobic (-NH-). It is noteworthy that the simplest bicyclomycin analogue 8h is totally devoid of antibacterial activity. Adding functionality to this basic nucleus in order of increasing complexity furnishes the structures shown below. These basic bicyclic nuclei have "0
2%
NK0
*
NHo
'0 b$!H
'0$ %o
o h & "0 H
H
H
SI
1-0 b
l>d
all been prepared for the first time by cerric ammonium (18) The excellent procedure of Yoshimura was employed: Yoshimura, J.; Yamaura, M.; Suzuki, T.; Hashimoto, H. Chem. Lett. 1983,1001. (19) We thank Dr. Hans Maag for providing a spectrum of compound12b(R1= Rz = R3 = R4 = H).
Williams, Armstrong, Dung Table 11. Minimal Inhibitory Concentration" (fig/mL) 1Oc (R, = biCH2Ph, cyclomyR2 = OH, cinRo R, = H) 21-7023 G- rods Pseudomonas aeruginosa 56 > 1000 >1000 >loo0 Proteus vulgaris lOlN >loo0 Escherichia coli 94 >loo0 250 250 Klebsiella pneumoniae 369 >loo0 Serratia marcescens SM >loo0 >loo0 Serratia sp. 101 >loo0 >1000 Acinetobacter calcoaceticus 1000 >loo0 PC13 G+ cocci Streptococcus faecium >1000 >1000 ATCC 8043 Staphylococcus aureus 82 >loo0 >1000 Micrococcus luteus PCI 500 >loo0 Bacillus megaterium 164 500 >loo0 G+ rods >1000 >1000 Bacillus sp. E Bacillus subtilis 558 250 >1000 250 Bacillus sp. T A >1000 Mycobacterium phlei 78 >loo0 1000 500 G+ filament Streptomyces cellulosae 097 500 molds Paecilonyces uarioti M16 >loo0 >1000 >loo0 >1000 Penicillium digitatum 0184 >loo0 yeasts >1000 Candida albicans 155 Saccharomyces cerevisiae >1000 >1000 90
" Lowest concentration still showing zone of inhibition by the agar-diffusion well method (serial dilutions up to 1000 wg/mL).
nitrate deprotection18 of the corresponding N,N'-bis(pmethoxybenzyl) derivatives as described in the Experimental Section. Both the 6-hydroxy-5-demethylene derivative 8i and the 6-deoxy-5-methylenederivative 10b lack antimicrobial activity at the maximum levels tested (1 mg/mL). Antimetabolite tests were carried out on minimal agar medium by the agar-diffusion well method. Table I1 provides the antimicrobial spectrum of the only active analogue, 1Oc (R, = CH2Ph,R2 = OH, R3 = H); all other compounds tested against the 20 microorganism screen (see Table 11) were inactive. Although the activity exhibited by the N-benzyl compound 1Oc was relatively weak, we were surprised to discover that this material displayed a different spectrum of activity t h a n bicyclomycin, showing Gram-positive inhibition against Micrococcus luteus, Bacillus megaterium, Bacillus subtilus, Bacillus sp. TA, and Streptomyces cellulosae. It should also be noted that all of the compounds tested are racemic and (presumably) the activity exhibited by 1Oc results from a single antipode. The absolute configuration of the active antipode for analogue 1Oc remains to be determined; the different spectrum of activity suggests that the naturally configured antipode is not necessarily the one displaying antimicrobial activity. The activity displayed by compound 1Oc when compared to the total lack of antimicrobial activity of the demethylene 8 and deoxy (loa and lob) analogues would seem to support the hypotheses outlined in Schemes I and I1 that the partnership of the C5 exo-methylene and C-6 hydroxyl group in this unique bicyclic structure are obligate, minimal structural requirements for antimicrobial activity. However, the lack of activity displayed by the corresponding lipophobic derivative 10d indicates that the solubility characteristics of these structures are very important. Compound 10d,which contains the complete structural nucleus of bicyclomycin but lacks the C-l'-C-3' trihydroxyisobutyl side chain, indicates the potential importance of this moiety for binding, chelation, or penetration. The Ciba-Geigy study as well as the diminished activity of the C-1'/C-2' acyl derivatives of bicyclomycin also points to the importance of the C1 ' 4 3 ' functionality. The demethylenebicyclomycin de-
Bicyclomycin Analogues
Journal of Medicinal Chemistry, 1985, Vol. 28, No. 6 737
(270 MHz) (Me2SO-d6,Me4Si) 6 1.729-1.754 (2 H, m), 1.895-1.930 (2 H, m), 3.631-3.724 (2 H, m), 3.816 (1H, br e.), 4.972 (1H, d, J = 4.73 Hz), 8.405 (1H, br s), 8.897 (1H, br s); IR (NaC1, neat) 1685,1445,1415,1105,1025cm-'; mass spectrum, m/e 170 (M', 16), 169 (lag),155 (2.3), 142 (4.6), 127 (14.4), 111 (13.3), 97 (20), 85 (25.7), 83 (26.2), 77 (100). Note: Additional,firm evidence for the assigned structure was obtained by treatment of this material with NaH/BrCH,Ph in MezSO to afford in 55% yield 8,10-dibenzyl-8,10-diaza-2-oxabicyclo[4.2.2]decane-7,9-dione(Sb, R1 = CHZPh; R2 = R3 = R4 = H; see ref 11 for spectroscopic and analytical data). 8,lO-Bis(p-methoxybenzyl)-8,10-diaza-6-hydroxy-2-oxabicyclo[4.2.2]decane-7,9-dione (8,R1 = CH2Ph-p-OCH3; Rz = OH; R3 = R4 = H).To a stirred solution of 8,10-bis(p-methoxybenzyl)-8,l0-diaza-2-oxabicyclo[4.2.2]decane-7,9-dione14 (Sc; 163mg, 0.39 mmol, 1.0 equiv) in THF (7.5 mL) containing HMPA (0.35 mL, 1.987 mmol, 5.0 equiv) at -78 OC was added a solution of LDA (0.51 mmol, 10 equiv) in THF (2.5 mL). While the dark-colored solution was stirred for 34 rnin at -78 "C, a stream of dry O2was bubbled through the solution for 5 min at -78 "C; the cooling bath was removed, and oxygen was bubbled through the mixture until the temperature had reached ambient. Several drops of HzO was added and the mixture was diluted with ether, poured into HzO, and extracted thoroughly with ether. The combined organic extracts were dried over anhydrous NaZSO4, filtered, evaporated, and separated by PTLC silica gel (eluted with 6% acetone in EtzO) to afford 89 mg (53% or 62.6% based on recovered starting material) of the pure bridgehead alcohol 8: mp 187-187.5 OC (recryst CHzC12/Etz0/hexanes); 'H NMR (270 MHz) (CDC13,CHC13)6 1.463-1.533 (2 H, m), 0.778-1.881 (2 H, m), 3.257 (1H, dd, J1= 13.32 Hz, Jz = 9.23 Hz), 3.714-3.723 (1H, m), 3.758 (3 H, s), 3.771 (3 H, s), 4.191 (1H, '/2AB q, J = 14.26 Hz), 4.503 (1H, '/zAB q, J = 13.88 Hz), 4.725 (1H, s), 4.777 (1H, '/zAB 4, J = 13.88 Hz), 4.925 (1H, '/2AB q, J = 14.26 Hz), 5.158 (1 H, s), 6.776-6.852 (4 H, m), 7.197-7.229 (2 H, m), Experimental Section 7.340-7.428 (2 H, m); IR (NaCl, neat) 3370,1675,1610,1505,1430, 1240, 1170, 1100, 1025, 920,800 cm-'; mass spectrum, m / e 426 'H NMR spectra were recorded on JEOL FX-100 (la0 MHz), (M', 3.6), 340 (l.O), 305 (2.1), 241 (11.3), 226 (10.9), 219 (198), IBM/Bruker WP-270 (270 MHz) and WP-200 (200 MHz), or 210 (1.4), 192 (10.2), 175 (2.1), 162 (492), 156 (16.9), 149 (7.3), 136 Nicolet (360 MHz) spectrometers and are reported in 6 values. (11.9), 121 (loo), 116 (97.9), 101 (39.6). Anal. (C&Z~N&) c , Melting points were recorded on a Mel-Temp instrument in open H, N. capillaries and are uncorrected. Microanalyses are within &0.4% 8,10-Diaza-6-hydroxy-2-oxabicyclo[ 4.2.21decane-7,g-dione of the calculated values. Infrared spectra were recorded on a (Si,R1 = H;Rz= OH;R3 = R4 = H). To a stirred, room temBeckman 4240 spectrophotometerand are reported as A- (cm-'). perature solution of 8 (R, = CHzPh-p-OMe,Rz = OH, R3 = R4 Low-resolution mass spectra were determined on a VG = H) (35 mg, 0.082 mmol, 1.0 equiv) in MeCN/H20 (1.1mL, 2:l MM16F-GC mass spectrometer. v/v) was added CAN (202 mg, 0.369 mmol, 4.5 equiv) in one Thin-layer chromatography (TLC) was carried out on E. Merck portion. The mixture was stirred for 80 rnin at room temperature 0.25-mm precoated silica gel glass plates (60F-254) by using 5% and directly separated on PTLC silica gel (eluted with 12% MeOH phosphomolybdic acid in ethanol-heat and/or UV light as dein CHZClz)to afford 5.2 mg (35%) of the deprotected amide Si veloping agent. Preparative-layer chromatography (PTLC) was as an amorphous white solid:21 'H NMR (270 MHz) (MezSO-d6, carried out on glass-backed TLC plates with a fluorescent indicator Me4Si)6 1.550-1.723 (2 H, m), 1.767-2.035 (2 H, m), 3.585-3.760 on a Harrison Research chromatotron using 1.0-, 2.0-, or 4.0-mm (2 H, m), 4.802 (1H, d, J = 4.37 Hz), 6.592 (1H, s), 8.706 (1H, layer thickness silica gel adsorbents. Separations of less than 50 br s), 8.983 (1H, d, J = 4.37 Hz); IR (NaC1, neat) 3260,3200,1685, mg were carried out on standard glass-backed E. Merck 0.25-mm 1440, 1275, 1130, 1070 cm-'. silica gel plates; the separated products were eluted from the S,l0-Dibenzyl-S,l0-diaza-6-hydroxy-2-oxabicyclo[ 4.2.21adsorbent with distilled THF. Flash column chromatographywas decane-7,g-dione(Sj, Rl = CH2Ph; R2 = OH; R, = R4 = H). performed by using Woelm silica gel 32-63. To a stirred solution of 8,10-dibenzyl-8,10-diaza-2-oxabicycloSolvents and reagents were all purified and dried according [4.2.2]decane-7,9-dione(Sb,R1 = CH2Ph; R2 = R3 = H; see ref to standard protocol. NMR multiplicities are reported by using 11for preparation) (25 mg, 0.07 mmol, 1.0 equiv) and HMPA (62 the following abbreviations: s, singlet; d, doublet; t, triplet; q, pL, 0.35 mmol, 5 equiv) in THF (2 mL) at -78 "C was added LDA quartet; m, multiplet; J,coupling constant in hertz. The chemical (0.107 mmol, 1.5 equiv) in THF (1 mL). After stirring of the shifts of protons part of an AB quartet ('/zAB q) was calculated dark-colored solution for 50 min at -78 OC, a stream of dry O2 by using a standard weighting formula. gas was bubbled through the solution for 70 min a t -78 OC, and 8,10-Diaza-2-oxabicyclo[4.2.2]decane-7,9-dione (Sh, R1= the mixture was quenched with H20 (0.1 mL). The reaction was R2 = R3 = R4 = H). To a stirred, room temperature suspension of 8,10-bis~p-methoxybenzyl)-8,l0-diaza-2-oxabicyclo[4.2.2]de-allowed to warm to room temperature, stirred 20 min, diluted with CHzCl2, poured into brine, and thoroughly extracted with CH2C12. cane-7,9-dione (Sc),prepared as described in ref 14 (68 mg, 0.165 The combined extracts were dried over anhydrous Na2S04,film o l , 1.0 equiv), in 33% aqueous acetonitrile (1.5 mL) was added tered, evaporated and separated on PTLC silica gel (eluted with CAN (318 mg, 0.58 mmol, 3.5 equiv). The mixture was stirred EhO)to afford the tertiary alcohol Sj (9.3 mg, 35.7% or 53% based at room temperature for 2 h and absorbed onto PTLC silica gel on recovered starting material) oil: 'H NMR (100 MHz) (CDCl,, (eluted with 10% MeOH in CHzC1z) to afford 16 mg (57%) of the debenzylated product Sa as an amorphous white solidz1 NMR
rivative 12c was also inactive, which again suggests that the complete structure of bicyclomycin is generally obligate for antimicrobial activity. The active analogue lOc, which displays a different spectrum of activity than bicyclomycin, is indicative of a distinct enzymic target; the mechanism of action in terms of chemical interaction with the bacterial proteins, however, may be similar for both 1Oc and 1. It is expected that demonstration of enzyme inhibitory properties for these compounds will be forthcoming. Preliminary evaluation of totally synthetic (&)-bicyclomycin16 showed half the antimicrobial activity of the optically pure (+) natural sample against E. coli 94 and Klebsiella pneumoniae 369. This result indicates that the enantiomorph in the racemate is devoid of antimicrobial activity. The bicyclo[3.2.2] homologues 9a-e, 17, and 18 did not exhibit antimicrobial activity, again indicating that this bicyclic nucleus lacks intrinsic activity. Attempts to hydroxylate 18 at the bridgehead positionz0 have thus far been unsuccessful in producing testable amounts of material. Thus a direct comparison of the bicyclo[3.2.2] homologue of 1Oc is presently not available and will have to await future scrutiny. These preliminary investigations into the pharmacological potentialities of a unique class of antibiotics first represented by 1 suggest many interesting structural and mechanistic experiments to further test the hypotheses outlined in Schemes 1-111 as well as fostering more refined notions of the chemical mechanism of action of bicyclomycin. Studies along these lines are in progress and shall be reported on in due course from these laboratories.
(20) Compound 18 can be regioselectively elaborated at both
bridgehead positions exactly analogous to 10a via generation and quenching of the corresponding bridgehead carbanions.
(21) Compounds reported as amorphous solids were recalcitrant to recrystallization and were precipitated from MeOH or THF;
combustion analytical data were not obtainable on these materials.
738 Journal of Medicinal Chemistry, 1985, Vol. 28, No. 6 CHC1,) 6 1.46-2.06 (4 H, m), 3.19-4.07 (2 H, m), 4.24 (1H, 1/2AB q, J = 14.4 Hz), 4.62 (1 H, l/zAB q, J = 13.9 Hz), 4.68 (1 H, s, D20 exch), 4.79 (1H, '/2AB q, J = 13.9 Hz), 5.04 (1H, l/zAB q, J = 14.4 Hz), 5.18 (1H, s), 7.30-7.47 (10 H, m); IR (NaC1, neat)
Williams, Armstrong, Dung
7,9-Dibenzyl-7,9-diaza-4-vinyl-2-oxabicyclo[ 3.2.21nonane6,8-dione (9d, R1= CH2Ph;Rz= R3 = H; R4 = CH==CH,). To a stirred solution of 16 (Bn = CHzPh) (110 mg, 0.289 mmol, 1.0 equiv) in THF (3 mL) at 0 "C were added Et,N (81 fiL, 0.58 mmol, 3400,3060,3015,1675,1600,1585,1495,1435,1260,1085,730, 2.0 equiv) and methanesulfonyl chloride (25 fiL, 0.31 mmol, 1.1 690 cm-'; mass spectrum, m / e 366 (M', 0.7), 275 (0.7), 57 (100). equiv). The mixture was stirred for 30 min, diluted with EtzO, 7,9-Bis(p -methoxybenzyl)-7,9-diaza-4-vinyl-2-oxabicyclo- filtered, evaporated, and purified by PTLC silica gel (eluted with 33% hexanes in EtOAc) to afford 130 mg (98.7%) of the corre[3.2.2]nonane-6,8-dione (9, R1= CHzPh-p-OCH,; Rz= R3 = sponding mesylate 9 (R1 = CHzPh, Rz = R, = H, R4 = H; R4 = CH=CH2) [note: the preparation of 7,9-bis(p-methCHzCHzOS02CH3): 'H NMR (100 MHz) (CDCl,, Me&) 6 oxybenzyl)-7,9-diaza-4-[ 2-(phenylselenyl)ethyl]-2-oxabicyclo1.33-1.92 (3 H, m), 2.96 (3 H, s), 3.24-4.18 ( 5 H, m), 4.36-4.87 [3.2.2]nonane-6,8-dione (9, R1 = CH2Ph-p-OMe; R2 = R3 = H; R4 = CH2CH2SePh)appem in ref 16bl: lH NMR (CDC13,Me4Si) (4 H, m), 5.11 (1H, s), 7.27-7.33 (10 H, m); IR (NaCl, neat) 1695, 6 1.20-1.40 (2 H, m), 2.20-2.38 (1 H, m), 2.72 (2 H, t, J = 7.80 1450, 1355, 1170, 950, 915, 725 cm-'. Hz), 3.51 (1 H, dd, J ~ c10.31 Hz, J,,, = 12.59 Hz), 3.75 (1H, To a stirred suspension of diphenyl diselenide (182 mg, 0.58 s), 3.78-3.88 (1H, m), 3.79 (3 H, s), 3.80 (3 H, s), 4.19 (1H, '/&3 mmol, 1.1equiv) in EtOH (4 mL) at 0 "C was added NaBH4 (46 q, J = 14.61 Hz), 4.29 (1 H, 1/2AB q, J = 14.86 Hz), 4.74 (1 H, mg, 1.22 mmol, 2.3 equiv). After the mixture was stirred for 10 '/2AB q, J = 14.86 Hz),4.74 (1H, 1/2ABq, J = 14.61 Hz), 5.02 rnin at 0 "C, a solution of the mesylate obtained as above (240 (1H, s), 6.83 (2 H, d, J = 8.64 Hz), 6.83 (2 H, d, J = 8.68 Hz), mg, 0.53 mmol, 1.0 equiv) in EtOH (2 mL) was added dropwise. 7.05 (2 H, d, J = 8.68 Hz), 7.10 (2 H, d, J = 8.64 Hz), 7.26-7.30 The mixture was stirred for 1.5 h, diluted with CHZClz,poured (3 H, m), 7.45-7.49 (2 H, m); IR (NaC1, neat) 1691, 1612, 1515, into brine, and thoroughly extracted with CH2ClP.The combined 1247,1030 cm-l; mass spectrum, m/e 580 (M', 5.2), 459 (3.5), 423 extracts were dried over anhydrous Na2SO4,filtered, and evap(2.4), 121 (100). orated to afford the crude selenide (310 mg), which was directly To a stirred solution of 9 (R, = CH2Ph-p-OMe,R2 = R3 = H, used for the next step without further purification. Treatment R4 = CHZCH2SePh)l6(80 mg, 0.138 mmol, 1.0 equiv) in THF (4 of this material in THF ( 5 mL) with 30% H,Oz (0.16 mL, 5.3 mL) at room temperature was added 30% HzOz(0.02 mL, 0.691 mmol, 10 equiv) at 0 "C (1min addition) was followed by warming mmol, 5.0 equiv), and the mixture was heated to reflux. After the reaction to ambient temperature. The mixture was stirred 20 min, the mixture was cooled to room temperature, diluted with 29 h at room temperature, refluxed for 1.5 h, cooled, diluted with CH2C12,poured into HzO, and exhaustively extracted with CHZCl2. CH2ClZ,poured into 0.1 N HC1, and thoroughly extracted with The combined extracts were dried over anhydrous Na2S04,filCH2Cl2 The combined organic extracts were dried over anhydrous tered, concentrated, and separated by PTLC silica gel to afford Na2S04,filtered, evaporated, and purified by PTLC silica gel 47 Wg (81%) of olefin 9 as a solid: mp 115-116 "C (recryst (eluted with 33% EtOAc in hexanes) to afford 71 mg (37.2%) of CH2C12);lH NMR (270 MHz) (CDCl,, (CH3)4Si)6 2.85 (1H, dd, the olefin 9d as colorless needles: mp 127.5-128.5 "C (recryst J ~=G7.52 Hz, Jvic= 7.12 Hz, JeC= 7.12 Hz), 3.69-3.89 (2 H, m), CH2Clz/hexanes);'H NMR (360 MHz) (CDCI,, CHC1,) 6 2.42-2.50 3.79 (6 H, s),3.88 (1H, s), 4.10 (1 H, 1/2ABq,J = 14.64 Hz), 4.29 (1H, m), 3.67 (1H, dd, J = 12.87 Hz, J = 12.87 Hz), 4.02 (1H, (1H, 1/2ABq, J = 14.74 Hz), 4.77 (1H, l/zAB q, J = 14.64 Hz), dd, J = 12.87 Hz, J = 12.87 Hz), 4.10 (1H, d, J = 2.77 Hz), 4.70 4.96 (1H, '/2AB q, J = 14.74 Hz), 5.07 (1H, s), 5.11 (1H, d, Jci, (1H, '/zAB 9, J = 14.50 Hz), 4.73 (1H, '/2AB q, J = 14.75 Hz), = 9.55 Hz), 5.15 (1 H, d, Jt,,, = 17.05 Hz), 5.23 (1 H, ddd, Jcia 5.02 (1H, l/zAB q, J = 14.50 Hz), 5.07 (1H, '/2AB q, J = 14.75 = 9.55 Hz, = 17.05 Hz, Jvi, = 7.52 Hz), 6.85 (4 H, d, J = 8.47 Hz), 5.16 (1 H, dd, J = 0.82 Hz, J = 17.54 Hz), 5.33 (1 H, d, J Hz), 7.12 (4 H, d, J = 8.47 Hz); IR (NaC1, neat) 1690, 1612, 1513, = 9.94 Hz), 5.38 (1H, s), 5.76 (1H, ddd, J = 7.55 Hz, J = 9.94 1240, 1030 cm-'; mass spectrum, m/e 422 (M', 7.1), 301 (11.2), Hz, J = 17.54 Hz), 7.02-7.40 (10 H, m); IR (NaCl,neat) 1690, 1500, C, H , N. 217 (2.51, 121 (100). Anal. (C24H26N205) 1450, 1215, 1075, 750 cm-'; mass spectrum, m/e 362 (M+, 8.2), 7,9-Diaza-4-vinyl-2-o~abicyclo[3.2.2]nonane-6,S-dione (9c, 332 (2.2), 292 (2.3), 271 (2.3), 264 (2.2), 91 (100). R1= Rz = R3 = H; R4 = CH=CH2). To a stirred solution of 7,9-Dibenzyl-7,9-diaza-4-( hydroxymet hyl)-2-oxabicyclo7,9-bis@-methoxybenzyl)-7,9-diaza-4-vinyl-2-oxabicyclo[ 3.2.21[3.2.2]nonane-6,8-dione (17, Bn = CH,Ph). A stream of ozone nonane-6,8-dione (9, R1 = CH2Ph-p-OCH3;Rz = R3 = H; R4 = was bubbled through a CH2Clz(3 mL) solution of 9d (R, = CHzPh, CH=CH2) (100 mg, 0.237 mmol, 1.0 equiv).in CH&N (0.48 mL) Rz = R3 = H, R4 = CH=CHz), (47 mg, 0.13 mmol, 1.0 equiv) for and H20 (0.24 mL) was added ceric ammonium nitrate (324.7 mg, 100 min. The mixture was allowed to come to room temperature, 0.592 mmol, 2.5 equiv). The mixture was allowed to stir for 15 evaporated, THF (3 mL) was added, and the mixture was cooled min at ambient temperature, diluted with CHZCl2,and immeto 0 O C . To this cold, stirred solution was added LAH (4 mg, 0.1 diately separated on chromatotron (PTLC) silica gel (eluted with mmol, 0.75 equiv). The mixture was stirred for 2 h, diluted with 10% MeOH in CH2Clz,then 20% MeOH in CH2C12)to afford CHZCl2,poured into 0.1 N HC1, and thoroughly extracted with unreacted starting material (49 mg), a mixture of mono-N-deCH2C12.The combined organic extracts were dried over anhydrous protected olefins (21 mg), and the desired N,N-deblocked olefin NaZSO4,filtered, evaporated, and purified by PTLC silica gel 9c ( 5 mg, 12% or 22% by conversion) as an amorphous solid.21 (eluted with 9% hexanes in EtOAc) to afford 11 mg (23%) of 9c: 'H NMR (270 MHz) (CD,OD, Me4%) 6 2.75 (1H, m), 3.54 alcohol 17: mp 161-162 "C (recryst CH,Clz/hexanes). 'H NMR (1 H, s), 3.60 (1 H, dd, J = 9.76, J = 12.76 Hz), 3.85 (1 H, dd, (100 MHz) (CDCl,, Me@) 6 1.74-2.00 (1 H, m), 3.33-4.11 ( 5 H, J = 5.44, J = 12.76 Hz), 4.70 (1 H, s), 5.13 (1 H, dd, J = 10.43, m), 4.35-4.89 (4 H, m), 5.10 (1H, s), 7.31 (10 H, s); IR (NaCl, neat) J = 1.13 Hz), 5.20 (1H, dd, J = 17.51, J = 1.13 Hz), 5.65 (1H, 3440, 1695, 1500, 1455, 1285, 1265, 1070, 905, 730 cm-'; mass dd, J = 10.43, J = 17.51, J = 7.23); IR (NaCl, neat) 3250, 1690, spectrum, m / e 366 (M+,7,1), 336 ( L l ) , 292 (12.4), 275 (2.4), 91 1615,1518,1245,1050cm-'. The mixture of mono-N-deprotected (100). olefins upon treatment with CAN (6 equiv) exactly as described The diastereomer 17 (epimeric at C-4) was obtained from the above led to ca. 50% yield of the desired compound 9c. 7,9-Dibenzyl-7,9-diaza-4-(2-hydroxyethyl)-2-oxabicyclo- diastereomericbicyclic alcohol 16 (epimericat C-4) in 53% overall yield following the same procedures described above. Spectro[3.2.2]nonane-6,8-dione (16, Bn = CHzPh). To a stirred solution scopic and analytical data is as follows for this series. of diol 1S16(670 mg, 1.36 mmol, 1.0 equiv) in T H F (20 mL) was 4-epi-17: mp 128-128.5 OC (recryst CH2C12/EtzO/hexanes); added silver perchlorate (566 mg, 2.73 mmol, 2.0 equiv) at 25 "C. 'H NMR (270 MHz) (CDC13, Me4Si) 6 1.665-1.695 (1 H, m), The mixture was allowed to stir 40 min, diluted with CHzC12, 2.298-2.425 (1H, m), 3.209 (1H, dd, J1= 10.751 Hz, J2 = 7.951 poured into 0.1 N NaOH, and thoroughly extracted with FH2ClZ. Hz), 3.397 (1H, dd, J1 = 10.751Hz, Jz = 5.843 Hz), 3.665 (1 H, The combined organic extracts were dried over anhydrous Na2S04, dd, 51 = 12.743 Hz, Jz = 9.122 Hz), 3.853 (1H, dd, J1 = 12.743 filtered, concentrated, and purified by PTLC silica gel (eluted Hz, JZ = 5.205 Hz), 4.110 (1H, d, J= 1.512 Hz), 4.344 (1H, '/zAB with 4% MeOH in CH2C12)to afford 510 mg (99%) of the bicyclic q, J = 15.042 Hz), 4.370 (1H, '/2AB q, J = 14.726 Hz), 4.871 (1 alcohol 16 (oil): 'H NMR (100 MHz) (CDCl,, Me4Si)6 1.26-1.92 H, l/zAB q, J= 15.042 Hz), 4.993 (1H, '/,AB q, J = 14.726 Hz), (3 H,m), 3.18-3.97 (5 H, m), 4.36-4.89 (4 H, m), 5.12 (1 H, s), 5.091 (1H, s), 7.138-7.474 (10 H, m); IR (NaC1, neat) 3430,3030, 7.30 (10 H, m); IR (NaC1, neat) 3480,1680,1455,1270,1220,1065, 1685,1495,1450,1355,1260,1160,1060,945,725,690cm-'. Anal. 750 cm-'; mass spectrum, m / e 380 (M', LO), 292 (3.0), 252 (1.11, (CziHzzNz04) C, H, N. 91 (49.5).
Bicyclomycin Analogues
Journal of Medicinal Chemistry, 1985, Vol. 28, No. 6 739
4 4 -7,9-Dibenzyl-7,9-diaza-4-vinyl-2-oxabicyclo[3.2.2]- into saturated NaCl (aqueous), and thoroughly extracted with CH2C12.The combined organic extracts were dried over anhydrous nonane-6,s-dione(9,R1= C H 2 P h ;Rz= R3 = H;R4 = CH= Na2S04,filtered, and evaporated, and the residue was separated CHz): mp 120-120.5 "C (recryst CHzClz/EhO/hexane); 'H NMR on PTLC silica gel (eluted with 50% ether in hexanes) to afford (270 MHz) (CDC13,Me4Si) 6 2.869-2.902 (1H, m), 3.465-3.899 the diol 12a (60.3 mg, 32.3% major diastereomer or 46.5% based (2 H, m), 3.918 (1H, d, J = 1.416 Hz), 4.217 (1H, l/zAB q, J = on recovered Sj; plus 15.3 mg of a diastereomer, 8.2% or 12% 14.889 Hz), 4.336 (1H, '/2AB 9, J = 14.990 Hz), 4.891 (1H, '/2AB based on recovced Sj) plus unreacted Sj (41.3 mg, 30.6%). q, J = 14.889 Hz), 5.074 (1H, '/2AB q, J = 14.990 Hz), 5.090 (1 Major diastereomer 12a: mp 168-169 "C (recryst Et20/ H, s), 5.133-5.408 (3 H, m), 7.184-7.346 (10 H, m); IR (NaC1, neat) 3060,3030,1680,1495,1450, 1425, 1270,1165, 1075,1060,910, hexanes); 'H NMR (360 MHz) (CDC13,CHC13) 6 0.738 (3 H, s), 1.140-1.367 (2 H, m), 1.330 (3 H, s), 1.337 (3 H, s), 1.683-1.767 725,690 cm-'. Anal. (CzzHz2N203) C, H, N. (1H, m), 2.033-2.133 (1H, m), 2.721-2.786 (1H, m), 3.449-3.518 8,10-Diaza-5-methylene-2-oxabicyclo[4.2.2]decane-7,9-dione (1 H, m), 3.748 (1 H, '/,AB q, J = 9.16 Hz), 4.097 (1H, '/,AB (lob,R1= Rz = R3 = H).To a stirred solution of 10l6 (R1 = q, J = 9.16 Hz), 4.603 (1H, d, J = 9.97 Hz), 4.611 (2 H, s), 4.693 CHzPh-p-OCH3,R, = R3 = H) (14 mg, 0.03 mmol, 1.0 equiv) in (1H, 1/2ABq, J = 15.19 Hz), 4.915 (1H, s, D20 exch), 5.145 (1 MeCN (0.1 mL) was added CAN (72.75 mg, 0.133 mmol, 4.0 H, l/zAB q, J = 15.19 Hz), 6.505 (1H, d, J = 9.97 Hz, DzO exch), equiv). The mixture was stirred for 1.2 h, diluted with MeOH 7.20-7.60 (10 H, m); IR (NaC1, neat) 3400,3060,3030,1665,1605, (1mL), and separated by PTLC silica gel (eluted with 20% MeOH 1500,1435,1405,1380,1250,1205,1075,905,850,730,700 cm-'. in CHC1,) to afford 5.5 mg (91%) of the fully deprotected bicyclic Anal. (C28H34N207) C, H, N. piperazinedione 10b as an amorphous solid plus 1 mg (ca. 8%) Minor diastereomer 12a: 'H NMR (100 MHz) (CDCl,, of a mixture of mono-N-(p-methoxybenzy1)piperazinediones(10): CHC13) 6 1.19-2.16 (13 H, m), 2.78-3.53 (2 H, m), 3.14 (1H, d, 'H NMR (270 MHz) (MezSO-d8,MezSO) 6 2.35-2.60 (2 H, m), J = 4.39 Hz), 3.65 (1H, '/2AB q, J = 8.54 Hz), 4.15 (1H, '/,AB 3.78 (1H, dd, J = 1.46 Hz, J = 6.84 Hz), 3.83 (1H, dd, J = 1.46 q, J = 8.54 Hz), 4.65 (2 H, s),4.79 (1H, d, J = 4.39 Hz, DzO exch), Hz, J = 6.84 Hz), 4.26 (1H, s), 4.87 (1H, s), 5.00 (1H, s), 5.06 (1 H, E), 8.68 (1H, DzO exch), 9.02 (1 H, D20 exch); IR (NaC1, 4.91 (1H, E, DzO exch), 4.94 (1H, 1/2ABq, J = 15.13 Hz), 5.42 (1H, 1/2AB q, J = 15.13 Hz), 7.20-7.59 (10 H, m); IR (NaCl, neat) neat) 3300-3200, 1680, 1620, 1250 cm-'. 3420,3060,3030,1660,1605,1495,1455,1400,1375,1325,1255, 8,10-Diaza-5-methylene-6-hydroxy-2-oxabicyclo[ 4.2.21de1100, 1055,905, 725 cm-'. (Note: The minor diastereomer was cane-7,g-dione(lOd,R1= H;Rz= OH;R3 = H).To a MeCN not submitted for bioassay.) (0.1 mL) solution of 10le(R1 = CH2Ph-p-OCH3,R2 = OH, R3 = S,lO-Bis(p-methoxybenzyl)-8,10-diaza-l-[2'-methyl1'H) (4.0 mg, 0.009 mmol, 1.0 equiv) was added HzO (0.01 mL) hydroxy-2',3'-(isopropylidenedioxy)propyl]-2-oxabicyclofollowed by CAN (22.53 mg, 0.041 mmol, 4.5 equiv). The reaction was stirred for 1.2 h at room temperature, diluted with MeOH [4.2.2]decane-7,9-dione(19,R1= CHzPh-p-OCH3; & = H;R3, (1 mL), and separated by PTLC silica gel (eluted with 5:l R4 = C(CH,),). To a stirred, -78 "C solution of Sc (R, = CH,Ph-p-OCH,, R2 = R3 = R4 = H) (101 mg, 0.246 mmol, 1.0 CHC13/MeOH) to afford 1.2 mg (66.4%) of the fully deprotected equiv) containing HMPA (0.21 mL, 1.23 mmol, 5.0 equiv) in THF amide 10d plus trace amounts of the monodeprotected mixture: mp 168-169 "C (recryst acetone/THF); 'H NMR (270 MHz) (4 mL) was added a solution of LDA (0.27 mmol, 1.1equiv) in THF (2 mL). The mixture stirred for 42 min at -78 "C and (Me2SO-d6,Me2SO) 6 2.50-2.70 (2 H, m), 3.56 (1H, dd, J,,, = chlorotrimethylsilane (47 pL, 0.369 mmol, 1.5equiv) was added. 9.10 Hz, Jvic 2.5 Hz), 3.78 (1H, dd, J v i c = 2.5 Hz, J,,, = 11.1 Hz), 6.95 (1H, s, D20 exch), 8.89 (1H, br s, DzO exch), 9.14 (1 The mixture was allowed to come to room temperature over a H, br s, DzO exch); IR (NaCl, neat) 3600-3200,1690,1620,1250 45-min period and was recooled to -78 "C. A solution of LDA cm-'. (0.61 mmol, 2.5 equiv) in THF (2 mL) was added and the solution stirred for 20 min at -78 "C and (*)-2,2,4-trimethy1-1,3-di7,9-Dibenzyl-7,9-diaza-4-met hylene-2-oxabicyclo[3.2.21oxolane-4-carboxaldehyde(177 mg, 1.23 mmol, 5 equiv) was added nonane-6,s-dione(18,Bn = CHzPh).A stirred solution of 17 (18.6 mg, 0.019 mmol, 1.0 equiv) was treated with methanesulfonyl dropwise. The mixture was allowed to come to room temperature. After stirring 35 min at room temperature, tetra-n-butylchloride (12 wL) and Et3N (29 ML)in THF (1mL) at 0 "C. After stirring at 0 "C for 30 min, the mixture was filtered and evapoammonium fluoride trihydrate (466 mg, 1.47 mmol, 6 equiv) was rated, 1,8-diazobicyclo[5.4.0]undec-7-ene(DBU) (6 pL, 0.38 mmol, added in one portion. After stirring 30 min at room temperature, the mixture was diluted with CH2C12,poured into brine, and 2.0 equiv) in toluene (1 mL) was added, and the mixture was thoroughly extracted with CH2C12 The combined extracts were heated at reflux for 17 h. After cooling to room temperature, the mixture was diluted with CH2C1,, poured into 0.1 N HCl solution, dried over anhydrous Na2S04,filtered, evaporated, and separated and thoroughly extracted with CH2C12. The combined organic on a silica gel column (50 g of silica gel, eluted with 5% MeOH extracts were dried over anhydrous Na2S04,filtered, evaporated, in CH2Cl,) to afford alcohol 19 (13 mg, 10% or 12% based on and separated on PTLC silica gel (eluted with 50% EtOAc in recovered starting material); other isomers (26.5 mg, 19.5% or hexanes) to afford 2.5 mg (38.5%) of olefin 18: mp 185-185.5 "C 24.2% based on recovered starting material) and starting material (recryst CHzClZ/Etz0/hexanes);'H NMR (270 MHz) (CDCl,, (19.5 mg, 19.3%) were also obtained. 'H NMR (270 MHz) (CDC13, Me4Si) 6 4.163 (1H, '/,AB q, J = 14.062 Hz), 4.335 (1H, 1/2AB Me4Si)6 1.199 (3 H, s), 1.370 (3 H, s), 1.409 (3 H, s), 1.748-1.886 q, J = 14.062 Hz), 4.363 (1H, s), 4.385 (1H,1/2ABq, J = 15.036 (2 H, m), 1.966-2.071 (2 H, m), 2.778-2.869 (1H, m), 3.483-3.574 Hz), 4.486 (1 H, l/zAB q, J = 14.741 Hz), 4.778 (1H, '/2AB q, (1H, m), 3.781 (3 H, s), 3.807 (3 H, s), 3.875-4.999 (5 H, m), 4.689 J = 14.741 Hz), 4.483 (1H, '/,AB q, J = 15.036 Hz), 5.069 (1H, (1 H, '/2AB q, J = 15.756 Hz), 6.655 (1 H, d, J = 9.571 Hz), 4.998 s), 5.106 (1H, s), 5.117 (1H, s), 7.200-7.361 (10 H, m); IR (NaCl, (1 H, l/zAB q, J = 15.756 Hz), 6.655 (1 H, d, J = 9.571 Hz), neat) 1695,1450,1215,1055,755cm-'; mass spectrum, m/e 348 6.753-6.889 (4 H, m), 7.179-7.266 (2 H, m), 7.486-7.538 (2 H, m); (M', 2.5), 261 (l.l), 243 (1.8),91 (100). Anal. (CzlHzoN203) C, IR (NaCl, neat) 3320,3050,1670,1610,1515,1435,1405,1300, H, N. 1245,1175,1105,1070,1030,905,730cm-l; mass spectrum, m/e An improved procedure for the preparation of 18 was found 544 (M', L4), 539 (l.l),497 (4.4), 441 (5.1), 121 (68.4), 59 (100). by converting the mesylate derived from 17 to the selenide folS,lO-Diaza-($'-met hyl-l',2',3'-trihydroxypropyl)-2-oxabilowed by HzOzoxidation and elimination (THF, reflux, 12 h) cyclo[4.2.2]decane-7,9-dione (12b,R1= R2 = R3 = R4 = H). exactly as described above for preparation of the vinyl derivative To a stirred, room-temperature solution of alcohol 19 obtained from 16 (77.4% overall yield from 17). above (10mg, 0.018 mmol, 1 equiv) in CHzClz(1mL) was added 8,10-Dibenzyl-8,10-diaza-6-hydroxy-l-[2'-methy~-l'-DMAP (24 mg, 0.2 mmol, 11equiv). After the mixture was stirred hydroxy-2',3'-(isopropylidenedioxy)propyl]-2-oxabicyclofor 10 min, trifluoroacetic anhydride (25 pL, 0.18 mmol, 10 equiv) [4.2.2]decane-7,9-dione(12a,R1= CH2Ph;R2 = OH;R,, R4 = was added. The mixture was allowed to stir 2 h and directly C(CH3),).To a stirred solution of alcohol S j (R, = CH,Ph, Rz separated on PTLC silica gel (eluted with 45% hexanes in EtOAc) = OH, R3 = R4 = H) (135 mg, 0.368 mmol, 1.0 equiv) in T H F (5 to afford the 1'-0-trifluoroacetate (oil) (8.4 mg, 72% or 90% based mL) at -100 "C was added n-BuLi (0.88 mmol, 2.4 equiv) dropwise. on recovered starting material) and starting material (2 mg, 20%): After the mixture was stirred for 15 min at -100 "C, 2,2,4-tri'H NMR (270 MHz) (CDC13,Me4Si)6 0.599 (3 H, s), 1.184 (3 H, methyl-1,3-dioxolane-4-carboxaldehyde (212 mg, 1.47 mmol, 4 s), 1.211 (3 H, s), 1.603-2.206 (4 H, m), 3.186-3.288 (1H, m), 3.524 equiv) was added. The mixture was allowed to stir 15 min at -100 (1 H, '/2AB q, J = 9.633 Hz), 3.791 (3 H, s), 3.804 (3 H, s), "C and 30 min at room temperature, diluted with CH2Clz,poured 3.810-5.051 (6 H, m), 4.396 (1H, 1/2ABq, J = 9.633 Hz), 6.145
740 Journal
of Medicinal Chemistry, 1985, Vol. 28,
No. 6
(1H, s), 6.775-7.595 (8 H, m); IR (NaC1, neat) 1785,1680,1610, 1510,1360,1300,1245, 1210,1170, 1145,1025,725 cm-l. To a stirred, room-temperature suspension of the trifluoroacetate (8.2 mg, 0.012 mmol, 1equiv) in MeCN/H20 (0.25 mL, 2:l v/v) was added CAN (41 mg, 0.072 mmol, 6 equiv) in one portion. The mixture was stirred for 2 h at room temperature and directly separated on PTLC silica gel (eluted with 20% MeOH in CH2Cl2)to afford triol 12b (1.5 mg, 45.5%) as an amorphous solid: mp 264 O C dec; ‘H NMR (270 MHz) (MezSO-d6,Me4Si) 6 1.155 (3H, s), 1.643-1.783 (2 H, m), 1.860-1.968 (2 H, m), 3.231-3.814 (5 H, m), 3.843 (1H, d, J = 7.714 Hz), 4.474 (1 H, br s, DzO exch), 5.166 (1H, d, J = 7.714 Hz, DzO exch), 5.176 (1H, s, DzO exch), 8.214 (1H, d, J = 3.466 Hz, D,O exch), 8.765 (1H, s, DzO exch); IR (NaC1, neat) 3340,1670,1600,1555,1540, 1415,1055, 1020 cm-’. d,lO-Bls(p -methoxybenzyl)-8,10-diaza-6-hydroxy-l-[2’met hyl-l’-hydroxy-2’,3’-(isopropylidenedioxy)propyl]-2-oxabicyclo[4.2.2]decane-7,9-dione (20, R1= CH,Ph-p -OCH3;R, = OH; R3, R4 = C(CH3),). To a stirred solution of alcohol 8 (R, = CH2Ph-p-OCH3,Rz = OH, R3 = R4 = H) (41 mg, 0.096 mmol, 1equiv) in THF (2 mL) at -100 OC was added n-BuLi (0.14 mL, 0.288 mmol, 3.0 equiv). After stirring of the mixture for 17 min (28 at -100 “C, 2,2,4-trimethyl-1,3-dioxolane-4-carboxaldehyde pL, 0.192 mmol, 2.0 equiv) was added and the mixture continued to stir at -100 “C for 42 min. The cooling bath was removed, allowing the temperature to reach ambient over a 40-min period. The mixture was diluted with CHZClz,poured into saturated NaCl solution, and thoroughly extracted with CH2C12. The combined organic extracts were dried over anhydrous NazS04,filtered, evaporated, and separated on PTLC silica gel (eluted with 10% hexanes in EtzO) to afford 22 mg (41%) of diol 20 and 12.2 mg (22.6%) of a diastereomer. Major diastereomer 2 0 ‘H NMR (270 MHz) (CDC13,Me4Si) 6 0.806 (3 H, SI, 1.352 (3 H, s),i.364 (3 H, e), 1.640-1.767 (2 H, m), 1.850-2.105 (2 H, m), 2.755-2.835 (1H, m), 3.490-3.585 (1 H, m), 3.733 (1H, ‘/,AB q, J = 8.37 Hz), 3.783 (6 H, s), 4.126 (1H, ‘/,AB q, J = 8.37 HZ), 4.586 (2 H, SI,4.606 (1H, d, J = i o Hz), 4.642 (1H, ‘/2AB 9, J = 15.31 Hz), 4.969 (1H, s), 5.091 (1 H, ‘/,AB q, J = 15.31 Hz),6.621 (1 H, d, J = 10 Hz), 6.810 (4 H, d, J = 8.6 Hz), 7.430 (2 H, d, J = 8.6 Hz), 7.507 (2 H, d, J = 8.6 Hz); IR (NaC1, neat) 3370,3300,3060,1655,1615,1515,1380, 1250,1175,1105,1070,1035,910,740 cm-’; mass spectrum, m/e 570 (M’, 0.4), 451 (1.7), 426 (l.l), 408 (4.6), 392 (0.6), 340 (3.4), 287 (lab),272 (3~1,233 (31,219(4.9),207 (2.1),192 (2.6),162(8.41, 148 (3.3), 136 (14.1, 121 (loo), 115 (23.4). Minor diastereomer 20 ‘H NMR (270 MHz) (CDCl,, Me4Si) 6 1.310 (3 H, S),i.391 (3 H, S),i.405 (3 H, SI, 1.608-1.775 (2 H, m), 1.880-2.085 (2 H, s), 2.810-2.915 (1 H, m), 3.182 (1 H, d, J = 4.26), 3.41-3.375 (2 H, m), 3.675 (1H, l/zAB q, J = 8.86 Hz), q, J = 8.58 HZ), 3.753 (3 H, s), 3.761 (3 H, SI, 3.708 (1H, I / ~ A B 3.957 (1H, ‘/,AB q, J = 8.58 HZ), 4.170 (1H, ‘/,AB q, J = 8.86 (1 H, I / ~ A B q, J = 13.2 HZ),4.948 (1H, SI, 5.405 (1 H ~ )4.604 , H, ‘/,AB q, J = 13.2 Hz), 6.75-6.821 (4 H, m), 7.365-7.486 (4 H, m), IR (NaC1, neat) 3430,3070,1665,1615,1520,1380,1245,1175, 1110, 1055, 1035,910,730 cm-’. 8,10-Diaza-6-hydroxy-( 2’-met hyl-l’,2’,3’-trihydroxypropyl)-2-oxabicyclo[4.2.2]decane-7,9-dione(12c, R1 = R3 = R4= H; R, = OH). To a stirred, room-temperature solution of diol 20 (R, = CHzPh-p-OCH3,Rz = OH, R3, R4 = C(CH3)z)(87 mg, 0.15 mmol, 1 equiv) in CHzClz (6 mL) was added 4-(dimethy1amino)pyridine (205 mg, 1.67 mmol, 11 equiv). After 15 min, tifluoroacetic anhydride (0.22 mL, 1.5 mmol, 10 equiv) was added. The reaction mixture was stirred at room temperature
Williams, Armstrong, Dung for 35 min, evaporated, and separated on PTLC silica gel (eluted with 25% hexane in EhO) to afford the 1’-0-trifluoroacetate (25 mg, 25%) and 1’,6-bis(O-trifluoroacetate)(57 mg, 50%). 1’-0-Trifluoroacetate: ‘H NMR (270 MHz) (CDC1, Me4Si) 6 0.359 (3 H, s), 1.127 (3 H, e), 1.146 (3 H, s), 1.506-1.726 (2 H, m), 1.829-2.028 (1H, m), 2.157-2.258 (1H, m), 3.047 (1H, ‘/,AB q , J = 9.433 Hz), 3.154-3.234 (1H, m), 3.709-3.885 (1H, m), 3.772 (3 H, s), 3.791 (3 H, s), 4.221 (1H, ‘/2AB 9, J = 9.433 Hz), 4.516 (1H, I / ~ A B 9, J = 13.237 HZ), 4.582 (1H, I / ~ A Bq, J = 14.804 Hz),4.597 (1H, ‘/2AB q, J = 13.327 Hz), 4.712 (1H, s, DzO exch), 5.024 (1H, ‘/,AB q, J = 14.804 Hz), 6.074 (1H, s), 6.784-6.884 (4 H, m), 7.381-7.645 (4 H, m); IR (NaCl, neat) 3350,1785, 1655, 1610, 1510, 1365, 1245,1210, 1165,1145,1030 cm-’. Exhaustive reacetylation of this compound produced the
1’,6-bis(O-trifluoroacetate). 1’,6-Bis( 0-trifluoroacetate): ‘H NMR (270 MHz) (CDC13, Me4Si) 6 0.360 (3 H, s), 1.125 (3 H, s), 1.144 (3 H, s), 1.497-1.703 (2 H, m), 1.937-2.071 (1H, m), 2.163-2.251 (1H, m), 3.047 (1 H, ‘/zAB q, J = 9.533 Hz), 3.134-3.243 (1H, m), 3.529-3.614 (1H, m), 3.769 (3 H, s), 3.788 (3 H, s), 4.221 (1H, ‘/,AB q, J = 9.533 Hz), 4.497-5.050 (4 H, m), 6.073 (1 H, s), 6.691 (4 H, m), 7.362-7.643 (4 H, m); IR (NaCl, neat) 1785, 1655, 1605,1505,1455, 1310, 1240,1205,1160, 1100, 1025 cm-’. To a stirred, room-temperature suspension of the 1’,6-bis(Otrifluoroacetate) obtained above (18 mg, 0.027 mmol, 1 equiv) in 0.5 mL of CH3CN/H20 (2/1, v/v) was added ceric ammonium nitrate (89 mg, 0.162 mmol, 6 equiv). The reaction mixture was stirred at room temperature for 2 h, evaporated, and separated twice on PTLC silica gel (1eluted with 25% EtOAc in EtzO; 2 eluted with 20% MeOH in CH,Cl,) to afford the tetrol 12c (3.7 mg., 47.4%) as a waxy solid. The same tetrol could also be obtained from the bis(0-trifluoroacetate) as described below. To a stirred, room temperature suspension of the bisacetate (19.5 mg, 0.025 mmol, 1 equiv) in 0.5 mL of CH3CN/H20(2/1, v/v) was added ceric ammonium nitrate (84 mg, 0.15 mmol, 6 equiv). The reaction mixture was stirred at room temperature for 2 h, evaporated, and separated twice on PTLC silica gel (1 eluted with 25% EtOAc in EtzO; 2 eluted with 20% MeOH in CHZClz)to afford 3 mg (41.4%) of tetrol12c: ‘HNMR (270 MHz) MezSO-d6, Me4Si) 6 1.151 (3 H, s), 1.617-1.757 (2 H, m), 1.803-2.029 (2 H, m), 3.343-3.531 (2 H, m), 3.671-3.777 (2 H, m), 3.850 (1H, d, J = 7.045 Hz), 4.430-4.452 (1H, m, DzO exch), 5.182 (1H, d, J = 7.045 Hz, D,O exch), 5.199 (1H, s, DzO exch), 6.534 (1H, br s, DzO exch), 8.542 (1H, s, DzO exch), 8.839 (1H, s, DzO exch); IR (NaC1, neat) 3320,1670,1390, 1115,1065, 1015 cm-’.
Acknowledgment is made to the National Institutes of Health (Grant lROl AIGM 18657), the donors of the Petroleum Research Fund, administered by the American Chemical Society, and the Colorado State University Biomedical Research Support (Grant 537276) for financial support of this work. NMR measurements at 360 MHz were obtained at the Colorado State University Regional NMR Center, funded by National Science Foundation Grant CHE 78-18581. We thank Fujisawa Pharmaceutical Co. for the generous gift of natural bicozamycin. We are especially indebted to Drs. David L. Pruess and Hans Maag of Hoffman-La Roche, Inc., for performing the antimicrobial tests. High-resolution mass spectra were obtained at the Midwest Center for Mass Spectrometry, a National Science Foundation Regional Instrumentation Facility (Grant No. CHE 8211164).
